Opiate receptors in brain have been identified biochemically by in vitro receptor binding assays and by opiate agonist-mediated adenylate cyclase. This study will utilize these techniques to explore the relationship between receptors and receptor-coupled membrane activities, such as receptor regulation and effector systems. The experiments have begun by examining properties of rat brain opiate receptors labeled by 3H-enkephalin analogs and 3H-opiate alkaloids. First, receptor binding of agonists is regulated by guanine nucleotides, and the interactions of opiate receptor binding sites and GTP-regulatory binding sites will be studied by separating receptors and regulators by ligand protection of sites from the actions of protein-modifying reagents. Specific sites responsible for GTP regulation will be studied by examining agonist effects on sub-populations of 3H-GTP binding sites in brain membranes and by solubilization of specific GTP-binding proteins. The relationship between proteins responsible for GTP regulation of agonist binding and GTP-mediated coupling of receptors and adenylate cyclase will be explored by labeling the coupling protein with cholera toxin-catalyzed 32P-NAD ribosylation. The effects of GTP on opiate actions will be studied by examining GTP-mediated coupling of opiate receptors to adenylate cyclase, the role of GTP in regulating multiple opiate receptors, and the relationship between GTP regulation and the development of tolerance. The lipid composition, and therefore fluidity, of membranes will be changed to study effects on both receptor binding and GTP regulation. The experiments in this proposal will involve in vitro assays of receptor binding and membrane enzyme assays, as well as techniques of membrane protein isolation and reconstitution. The overall goal is to characterize post-receptor events in opioid actions.